The metabolic processes commonly involved in the biotransformation of xenobiotics have been classified into functionalization reactions (phase I reactions), in which lipophilic compounds are modified via monooxygenation, dealkylation, reduction, aromatization, or hydrolysis. These modified molecules can then be substrates for the phase II reactions, often called conjugation reactions, as they conjugate a functional group with a polar, endogenous compound. Drug glucuronidation, a major phase II conjugation reaction in the mammalian detoxification system, is catalyzed by the UDP-glucuronosyltransferases (UGTs) (Batt AM, et al. (1994) Clin Chim Acta 226:171-190; Burchell et al. (1995) Life Sci. 57:1819-31).
The UGTs are a family of enzymes that catalyze the glucuronic acid conjugation of a wide range of endogenous and exogenous substrates including phenols, alcohols, amines and fatty acids. The reactions catalyzed by UGTs permit the conversion of a large range of toxic endogenous/xenobiotic compounds to more water-soluble forms for subsequent excretion (Parkinson A (1996) Toxicol Pathol 24:48-57).
The UGT isoenzymes are located primarily in hepatic endoplasmic reticulum and nuclear envelope (Parkinson A (1996) Toxicol Pathol 24:48-57), though they are also expressed in other tissues such as kidney and skin. UGTs are encoded by a large multigene superfamily that has evolved to produce catalysts with differing but overlapping substrate specificities. Three families, UGT1, UGT2, and UGT8, have been identified within the superfamily. UGTs are assigned to one of the subfamilies based on amino acid sequence identity, e.g., UGT1 family members have greater than 45% amino acid sequence identity (Mackenzie et al. 1997) Pharmacogenetics 7:255-69).
The UGT1 locus is located on chromosome 2q37, and contains at least 12 promoters/first exons, which are apparently able to splice with common exons 2 through 5, producing gene products having strikingly different N-terminal halves (amino acid sequence identities ranging from 24% to 49%), but identical C-terminal halves (FIG. 1). At least eight different isoenzymes are encoded by the UGT1 locus; at least one or more first exons encode pseudogenes. The different N-terminal halves encoded by the first exons confer different substrate binding specificities upon the UGT1 isoenzymes, while exons 2-5, which are present in all UGT1 isoenzyme mRNAs, encode the UDP-glucuronic acid binding domain, membrane anchorage site, and ER retention signal that are common to all UGT proteins (Ritter et al. (1992) J Biol Chem 267:3257-3261). UGT1 locus isoenzymes are best known for their role in glucoronidation and metabolism of many substrates, including bilirubin (1A1, 1 D1), planar and non-planar phenols, naphthols (1F1) (Ouzzine M, et al. (1994) Arch Biochem Biophys 310:196-204), anthraquinones, flavones, aliphatic alcohols, aromatic carboxylic acids, and steroids (Ebner T, et al. (1993) Drug Metab Dispos 21:50-55).
In addition to UGT1 exon usage, metabolism of endogenous and exogenous substrates can also be affected by competitive binding phenomena. For example, in some cases exogenous substrates for the UGT1 enzymes have a higher binding affinity or avidity for the enzyme than the endogenous UGT1 substrates. For example, UGT1*1, the major bilirubin-metabolizing form of UGT1, more readily binds both octyl-gallate and emodin than it binds bilirubin, thus indicating the potential of these xenobiotics to cause jaundice by inhibition of bilirubin binding to UGT1*1 (where 1*1 indicates that the first exon is used in the spliced gene product). UGT1*1 is also responsible for glucuronidation of the oral contraceptive ethinylestradiol (Ebner et al. (1993) Mol. Pharmacol. 43:649-54), and can also glucuronidate phenols, anthroquinones, flavones, and certain endogenous steroids.
As noted above, the first exon present in UGT1 can affect substrate binding specificity of the UGT1 gene product (for a review, see Burchell (1995) Life Sci. 57:1819-31). For example, UGT1*2 accepts a wide range of compounds as substrates including non-planar phenols, anthraquinones, flavones, aliphatic alcohols, aromatic carboxylic acids, steroids (4-hydroxyestrone, estrone) and many drugs of varied structure (Ebner et al. (1993) Drug. Metab. Disp. 21:50-5; Burchell (1995) Life Sci. 57:1 819-31). In contrast, UGT1*6 exhibits only limited substrate specificity for planar phenolic compounds relative to other human UGTs.
Polymorphisms can markedly affect binding of the endogenous substrate, which can be manifested as clinical syndromes. At least two conditions, Crigler-Najjar syndrome and Gilbert syndrome, are associated with UGT1 polymorphisms. Both of these syndromes are hereditary and are associated with predominantly unconjugated hyperbilirubinemia. Crigler-Najjar syndrome is associated with intense, persistent jaundice which begins at birth. Some affected infants die in the first weeks or months of life with kernicterus; others survive with little or no neurologic defect. Crigler-Najjar syndrome is caused by a defect in the ability of UGT1 to catalyze UDP-glucuronidation of bilirubin, resulting in accumulation of bilirubin in the blood (Erps et al. (1994) J. Clin. Invest. 93:564-70). Gilbert syndrome is a benign mild form of unconjugated hyperbilirubinemia that is characterized by normal liver function tests, normal liver histology, delayed clearance of bilirubin from the blood, and mild jaundice that tends to fluctuate in severity. As with Crigler-Najjar syndrome, Gilbert syndrome is associated with a defect in UGT1. Specific UGT polymorphisms that are known to be associated with disease are indicated in FIG. 1.
Alteration of the expression or function of UGTs may also affect drug metabolism. For example, there may be common polymorphisms in the human UGT1 gene that alter expression or function of the protein product and cause drug exposure-related phenotypes. Thus, there is a need in the field to identify UGT1 polymorphisms in order to provide a better understanding of drug metabolism and the diagnosis of drug exposure-related phenotypes.
Genbank accession number M84122 provides UGT1 exon 2, M84123 provides exons 3 and 4, M84124 provides 5, M84125 provides exon 1A, M84127 provides exon 1C, M84128 provides exon 1D, M84129 provides exon 1E, M84130 provides exon 1F, U39570 provides exon 1G, U42604 provides exon 1H, U39550 provides exon 1J.
The UGT gene superfamily and recommended nomenclature for describing UGT genes and alleles are reviewed in Mackenzie et al. (1997) Pharmacogenet. 7:255-69.
The two UGT1A6 genetic polymorphisms are described in Ciotti et al. (1997) Am. J. Hum. Genet. 61(Supp):A249. The identification of Asp446 as a critical residue in UGT1 is described in Iwano et al. (1997) Biochem. J. 325:587-91.
A review of the substrate specificity of human UDP-glucuronosyltransferases is provided by Burchell et al. (1995) Life Sci. 57:1819-31. For a review of drug glucoronidation in humans, see Miners et al. (1991) Pharmacol. Ther. 51:347-69.
At least twelve UGT1A1 polymorphisms have been identified and linked to disease. These UGT1A1 alleles, each of described in OMIM Entry 191740 and in OMIM Entry 143500 include:
1) UGT1*FB (UGT1A1, 13-BP DEL, EX2; 191740.0001), which contains a 13 bp deletion in exon 2 and is associated with Crigler-Najjar syndrome type I (CN-I);
2) UGT1A1, EXON4, C-T, SER-PHE (191740.0002), which contains a C-to-T transition in exon 4 (resulting in an amino acid change from serine to phenylalanine) is associated with CN-I and deficiency of both bilirubin-UGT and phenol-UGT activities in the liver;
3) UGT1A1, GLN331TER (191740.0003), which contains a C-to-T transition 6 bp upstream from the 3-prime end of exon 2 of the common region (replacement of a glutamine codon with a stop codon), is associated with CN-I;
4) UGT1A1, ARG341TER (191740.0004), which contains a nonsense mutation (CGA-to-TGA) in exon 3 and is associated with CN-I and a total absence of all phenol/bilirubin UGT proteins and their activities in liver homogenate by enzymologic and immunochemical analysis;
5) UGT1A1, GLN331ART (191740.0005), which contains an A-to-G transition 5 bp upstream of the exon 2/intron 2 boundary (resulting in a glutamine-to-arginine substitution), is associated with Crigler-Najjar Syndrome, type II (CN-II);
6) UGT1A1, PHE170DEL (191740.0006), which contains a deletion of the phenylalanine codon at position 170 in exon 1, and is associated with CN-I;
7) UGT1A1, SER376PHE (191740.0007), which contains a C-to-T transition in codon 376 (resulting in a change of serine to phenylalanine) and is associated with CN-I;
8) UGT1A1, GLY309GLU (191740.0008), which contains a G-to-A transition in codon 309 (resulting in a glycine to glutamic acid change) and is associated with CN-I;
9) UGT1A1, NT840, C-A, CYS-TER (191740.0009), which contains a C-to-A transversion at base position 840 in exon 1 (resulting in replacing a cysteine with a stop codon), is associated with CN-I;
10) UGT1A1, PRO229GLN (191740.00010), which contains C-to-A transversion at nucleotide 686 (changing proline-229 to glutamine), is associated with Gilbert syndrome;
11) UGT1A1, 2-BP INS, TA INS, TATAA ELEMENT (191740.00011) contains 2 extra bases (TA) in the TATAA element of the 5-prime promoter region of the gene (where normally an A(TA)6TAA element is present between nucleotides xe2x88x9223 and xe2x88x923) and is associated with Gilbert syndrome; and
12) UGT1A1, 1-BP INS, 470T INS (191740.00012), which contains 470insT mutation in exon 1 and is associated with CN-I.
Genetic sequence polymorphisms are identified in the UGT1 gene. Nucleic acids comprising the polymorphic sequences are used in screening assays, and for genotyping individuals. The genotyping information is used to predict an individuals"" rate of metabolism for UGT1 substrates., potential drug-drug interactions, and adverse/side effects.
Accordingly, in one aspect the invention features an isolated nucleic acid molecule comprising a UGT1 sequence polymorphism of SEQ ID NOS:87-124, as part of other than a naturally occurring chromosome. In related aspects, the invention features nucleic acid probes for detection of UGT1 locus polymorphisms, where the probe comprises a polymorphic sequence of SEQ ID NOS:87-124.
In another aspect the invention features an array of oligonucleotides comprising two or more probes for detection of UGT1 locus polymorphisms, where the probes comprise at least one form of a polymorphic sequences of SEQ ID NOS:87-124.
In still another aspect, the invention features a method for detecting in an individual a polymorphism in UGT1 metabolism of a substrate, where the method comprises analyzing the genome of the individual for the presence of at least one UGT1 polymorphism of SEQ ID NOS:87-124; wherein the presence of the predisposing polymorphism is indicative of an alteration in UGT1 expression or activity.
In one embodiment, the analyzing step of the method is accomplished by detection of specific binding between the individual""s genomic DNA with an array of oligonucleotides comprising two or more probes for detection of UGT1 locus polymorphisms, where the probes comprise at least one form of a polymorphic sequence of SEQ ID NOS:87-124.
In other embodiments of the method, the alteration is UGT1 expression or activity is tissue specific, or is in response to a UGT1 modifier. The UGT1 modifier may either induce or inhibit UGT1 expression.